To investigate biological processes that are critical to the progression of cancer and other diseases we use quantitative Single Molecule Localization Microscopy (qSMLM). qSMLM is a fluorescence-based imaging technique that evaluates single molecules with nanoscale precision. In typical qSMLM experiments, target molecules are detected with fluorescent reporters like optical highlighter proteins or antibodies labeled with photoswitchable dyes. Since these imaging agents have intricate photophysical properties, one major challenge in the field has been relating the number of localizations of reporters to the detected number of target proteins. To address this, we recently developed a new Surface Assay for Molecular Isolation (SAMI), that allows us to robustly count individual molecules. We use SAMI and other methodological advancements to study important biological mechanisms, aid in development of novel therapeutic agents, and advance diagnostics.

qSMLM for imaging of patient tissues

Tobin, Wakefield, et al., Sci Rep. 2018, 11, 15154

We apply advanced qSMLM methods to quantify both the density and nano-organization of membrane receptors in cells and in tissues ex vivo. We are focusing on assessing G-protein coupled receptors and receptor tyrosine kinases. Recently, we used qSMLM to quantify the organization of human epidermal growth factor receptor 2 (HER2), a receptor overexpressed in 20% of breast cancers and a target for immunotherapy. We combined tissue touch preparation with qSMLM (touch prep-qSMLM) to assess human tumor samples. We developed a straightforward analytical protocol and algorithms designed for rapid data analysis. These advancements allowed us to obtain quantitative results for tumor HER2 status within one day. We observed a significant positive correlation between touch prep-qSMLM and standard clinical HER2 screening methods which typically take 3-10 days. In addition to utility in patient diagnosis, we envision that the molecular details on density and organization of receptors within signaling domains may ultimately help clarify therapy resistance.

qSMLM for imaging of extracellular vesicles (EVs)

Both healthy and disease cells continuously shed membrane encapsulated particles called extracellular vesicles (EVs). EVs regulate intercellular communications through their cargo, which includes nucleotides, metabolites, lipids, and proteins. EVs contain a wealth of biological information and can reflect disease pathology. Moreover, EVs can be collected frequently and non-invasively from almost any accessible biofluid, such as blood, saliva, and urine. EVs are thus an attractive target for the early detection and monitoring of various diseases.

Patient biofluids contain complex mixtures of EVs; they originate from all tissues. A major challenge has been the detection of disease-specific EVs or tissue-specific EVs within the background of ‘other’ EVs. We have developed new methodology to isolate and characterize EV populations enriched in receptors that have high expression in either specific cell types or disease. Our qSMLM approach uniquely allows us to assess both the size and the molecular content of individual EVs. Since limited information on EVs is currently available at the molecular level, we expect these studies to help address EV heterogeneity and ultimately advance EVs as biomarker sources in a clinical setting.

qSMLM for imaging of nucleocytoplasmic transport

The nucleus and cytoplasm communicate through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope. NPCs are selectively permeable to certain macromolecules and thus regulate nucleocytoplasmic transport. To efficiently regulate selective transport, each NPC can shuttle thousands of molecules every second. Errors in this high throughput process can lead to large scale cellular dysregulation observed in a number of diseases, including neurodegenerative disorders and solid tumors. Despite these high stakes, resolution of specific mechanism(s) of transport has been challenging. To advance understanding of these mechanisms, we apply qSMLM to obtain single molecule data on NPC barrier mimics and on NPCs from intact nuclei. The resolution of nucleocytoplasmic transport mechanisms may ultimately provide novel targets and opportunities for drug development.